The crystal structure of full-length T7 DNA polymerase in complex with its processivity factor thioredoxin and double-stranded DNA in the polymerization active site exhibits two novel structural motifs in family-A DNA polymerases: an extended β-hairpin at the fingers subdomain, that interacts with the DNA template strand downstream the primer-terminus, and a helix-loop-helix motif (insertion1) located between residues 102 to 122 in the exonuclease domain. The extended β-hairpin is involved in nucleotide incorporation on substrates with 5′-overhangs longer than 2 nt, suggesting a role in stabilizing the template strand into the polymerization domain. Our biochemical data reveal that insertion1 of the exonuclease domain makes stabilizing interactions that facilitate proofreading by shuttling the primer strand into the exonuclease active site. Overall, our studies evidence conservation of the 3′–5′ exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating β-hairpin guides the template-strand into the polymerization active site after the T7 primase-helicase unwinds the DNA double helix ameliorating the formation of secondary structures and decreasing the appearance of indels.

中文翻译：

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T7 DNA 聚合酶开放构象的结构揭示了在聚合活性位点调节引物模板稳定性的新结构特征

全长 T7 DNA 聚合酶的晶体结构与其合成因子硫氧还蛋白和聚合活性位点中的双链 DNA 复合，在家族 A DNA 聚合酶中表现出两个新的结构基序：手指亚域处的扩展 β-发夹，即与引物末端下游的 DNA 模板链以及位于外切核酸酶结构域中残基 102 至 122 之间的螺旋-环-螺旋基序（插入 1）相互作用。延伸的 β-发夹参与核苷酸掺入具有 5'-突出端长于 2 nt 的底物，表明其在稳定模板链进入聚合结构域中的作用。我们的生化数据显示，外切核酸酶结构域的插入 1 产生稳定的相互作用，通过将引物链穿梭到外切核酸酶活性位点来促进校对。总体而言，我们的研究证明了 A 族 DNA 聚合酶之间 3'-5' 核酸外切酶结构域折叠的保守性，并突出了 T7 DNA 聚合酶的模块化结构。我们的数据表明，在 T7 引物解旋酶解开 DNA 双螺旋后，插入的 β-发夹引导模板链进入聚合活性位点，从而改善二级结构的形成并减少插入缺失的出现。